Method of nucleic acid sequence selection

ABSTRACT

The present invention provides a method for the rapid isolation and recovery of a desired target DNA or RNA molecules from a mixture or library containing such molecules. The method involves the use of biotinylated probes and enzymatic repair-cleavage to eliminate undesired library members from a sample.

FIELD OF THE INVENTION

The invention relates to an improved method for isolating and recoveringtarget DNA or RNA molecules having a desired nucleotide sequence.Specifically, it relates to a method for the rapid isolation of specificnucleic acid target molecules.

BACKGROUND OF THE INVENTION

The ability to clone gene sequences has permitted inquiries into thestructure and function of nucleic acids, and has resulted in an abilityto express highly desired proteins, such as hormones, enzymes,receptors, antibodies, etc., in diverse hosts.

The most commonly used methods for cloning a gene sequence involve thein vitro use of site-specific restriction endonucleases, and ligases. Inbrief, these methods rely upon the capacity of the "restrictionendonucleases" to cleave double-stranded DNA in a manner that producestermini whose structure (i.e. 3' overhang, 5' overhang, or blunt end)and sequence are both well defined. Any such DNA molecule can then bejoined to a suitably cleaved vector molecule (i.e. a nucleic acidmolecule, typically double-stranded DNA, having specialized sequenceswhich permit it to be replicated in a suitable host cell) through theaction of a DNA ligase. The gene sequence may then be duplicatedindefinitely by propagating the vector in a suitable host. Methods forperforming such manipulations are well-known (see, for example, Perbal,B. A Practical Guide to Molecular Cloning, John Wiley & Sons, N.Y.,(1984), pp. 208-216; Maniatis, T., et al. (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1982); Old, R. W. et al., In: Principles of Gene Manipulation, 2^(nd)Ed., University of California Press, Los Angeles, (1981), all hereinincorporated by reference).

In some cases, a gene sequence of interest is so abundant in a sourcethat it can be cloned directly without prior purification or enrichment.In most cases, however, the relative abundance of a desired target DNAmolecule will require the use of ancillary screening techniques in orderto identify the desired molecule and isolate it from other molecules ofthe source material.

A primary screening technique involves identifying the desired clonebased upon its DNA sequence via hybridization with a complimentarynucleic acid probe. In situ filter hybridization methods areparticularly well known (see, Sambrook, J., et al., In: MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)). In such methods, bacteria arelysed on the surface of the membrane filter and then incubated in thepresence of a detectably labelled nucleic acid molecule whose sequenceis complimentary to that of the desired sequence. If the extractcontains the desired sequence, hybridization occurs and thereby bindsthe labelled molecule to the adsorbent surface. The detection of thelabel on the adsorbent surface reveals that the bacteria sampledcontained the desired cloned sequence.

Although these screening methods are useful and reliable, they requirelabor-intensive and time consuming steps such as filter preparation andmultiple rounds of filter hybridization and colony platings/phageinfections. Generally, these procedures will screen up to 10⁶ colonieseffectively, but may take weeks or months to yield the desired clone.

Recently, other approaches have been developed to isolate recombinantmolecules which have eliminated the tedious filter-handling procedure.These approaches employ conventional hybridization technology coupledwith chromatography or magnetic particle technology. Rigas, B. et al.,for example, reported a method for isolating one plasmid species from amixture of two plasmid species. In the disclosed method, circulardouble-stranded plasmid DNA is hybridized to a RecA protein-coatedbiotinylated probe to form a stable triple-stranded complex, which isthen selectively bound to an agarose-streptavidin column (Rigas, B. etal., Proc, Natl. Acad. Sci. (U.S.A.) 83: 9591-9595 (1986)). The methodthus permits the isolation of cloned double-stranded molecules withoutrequiring any separation of the strands.

A DNA isolation method, termed "triplex affinity capture," has beendescribed in which a specific double-stranded genomic DNA is hybridizedto a biotinylated homopyrimidine oligonucleotide probe to form a"triplex complex," which can then be selectively bound tostreptavidin-coated magnetic beads (Ito, T. et al., Nucleic Acids Res.20: 3524 (1992); Ito, T. et al., Proc. Natl. Acad, Sci. (U.S.A.) 89:495-498 (1992)). Takabatake, T. et al. have described a variation ofthis technique that employs a biotinylated purine-rich oligonucleotideprobe to detect and recover the desired nucleic acid molecule(Takabatake, T. et al., Nucleic Acid Res. 20: 5853-5854 (1992)). Apractical drawback with these particular approaches is that they arerestricted to isolation of target DNA sequences containinghomopurine-homopyrimidine tracts.

Fry, G. et al. discuss a method for sequencing isolated M13-LacZphagemids (Fry, G. et al., BioTechniques 13:124-131 (1992)). In thismethod, a clone is selected and the phagemid DNA is permitted tohybridize to a biotinylated probe whose sequence is complementary to thephagemid's lacZ region. The biotinylated probe is attached to astreptavidin-coated paramagnetic bead. Since the DNA bound to the beadcan be separated from unbound DNA, the method provides a means forseparating the cloned sequence from the bacterial sequences that areinevitably present (Fry, G. et al., BioTechniques 13: 124-131 (1992)).

Still another method of screening recombinant nucleic acid molecules isdescribed by Kwok, P. Y. et al. This method, which is an extension ofPCR-based screening procedures uses an ELISA-basedoligonucleotide-ligation assay (OLA) to detect the PCR products thatcontain the target source (Kwok, P. Y. et al., Genomics 13: 935-941(1992)). The OLA employs an "reporter probe" and aphosphorylated/biotinylated "anchor" probe, which is captured withimmobilized streptavidin (Landegren, U. et al., Science 241:1077-1080(1988)).

The isolation of target DNA from a complex population using asubtractive hybridization technique has also been described (Lamar, E.E. et al., Cell 37:171-177 (1984); Rubenstein, J. L. R. et al., NucleicAcids Res. 18:4833-4842 (1990); Hedrik, S. M. et al., Science308:149-153 (1984); Duguid, J. R. et al., Proc. Natl. Acad. Sci.(U.S.A.) 85:5738-5742 (1988)). In such "subtractive hybridization"screening methods, the cDNA molecules created from a first population ofcells is hybridized to cDNA or RNA of a second population of cells inorder to "subtract out" those cDNA molecules that are complementary tonucleic acid molecules present in the second population and thus reflectnucleic acid molecules present in both populations

The method is illustrated by Duguid, J. R. et al. (Proc. Natl. Acad.Sci. (U.S.A.) 85:5738-5742 (1988)) who used subtractive hybridization toidentify gene sequences that were expressed in brain tissue as a resultof scrapie infection. A cDNA preparation made from uninfected cells wasbiotinylated and permitted to hybridize with cDNA made from infectedcells in a sample. Sequences in common hybridized to one another, andwere removed from the sample through the use of a biotin-binding(avidin) resin.

Weiland, I. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 87:2720-2724 (1990))reported an improved method of subtractive hybridization in which testerDNA was cleaved with a restriction endonuclease, and then permitted tohybridize to sheared driver DNA at high C₀ t values ("C₀ t" is theproduct of the initial concentration of DNA and the time of incubation).By cloning the double-stranded, PCR-amplified, unique DNA molecules intoa plasmid vector, it was possible to obtain an enrichment in therelative proportion of target sequences recovered.

Rubenstein, J. L. R. et al. (Nucleic Acids Res. 18:4833-4842 (1990))reported a further improvement in subtractive hybridization methods thatemployed single-stranded phagemid vectors to provide both the target andtester DNA. In the method, hybridized phagemid DNA-biotinylated driverstrand complexes are separated from unhybridized DNA by the addition ofstreptavidin. Unhybridized single-stranded DNA was subsequentlyconverted to the double-stranded form using Taq DNA polymerase and anoligonucleotide complementary to a common region found within thesingle-stranded DNA. The use of this method is, however, limited by theneed to follow a rigorous single-stranded phagemid purification protocolin order to obtain a preparation virtually free of contaminantdouble-stranded DNA. (Rubenstein, J. L. R. et al., Nucleic Acids Res.18:4833-4841 (1990)).

In sum, methods for isolating particular target nucleic acid moleculesare restricted by the abundance of the DNA target sequence, and bytime-consuming steps. Accordingly, a method that would expedite theisolation of desired target nucleic acid molecules and that could yieldessentially pure target DNA would be highly desirable.

SUMMARY OF THE INVENTION

The present invention provides a method for rapidly isolating nucleicacid molecules having a desired nucleotide sequence from other undesirednucleic acid molecules. Significantly, the present invention furtherrelates to an improved method of screening target nucleic acid moleculesemploying hybridization methodology combined with ligand separation, DNArepair, and restriction enzyme digestion technology.

In detail, the invention provides a method for recovering a desiredtarget nucleic acid molecule from a sample containing a mixture orlibrary of single-stranded nucleic acid containing the molecule, whereinthe method comprises the steps:

A. incubating the sample containing the nucleic acid mixture or libraryin the presence of a primer nucleic acid molecule complementary to asequence of the desired target molecule; the incubation being underconditions sufficient to permit the template-dependent extension of theprimer to thereby generate a double-stranded desired target molecule;

B. transforming single-stranded and double-stranded members of themixture or library into a host cell, and

C. recovering the desired molecule from the cell.

The invention additionally provides the embodiment wherein prior tocommencing step A, the method comprises the presteps:

(1) incubating an initial sample containing the nucleic acid mixture orlibrary in the presence of a haptenylated nucleic acid probe molecule,the probe molecules having a sequence complimentary to a nucleotidesequence of the desired target molecule; the incubation being underconditions sufficient to permit the probe to hybridize to the desiredtarget molecule and to thereby generate a hybridized molecule whereinthe target molecule is bound to the probe;

(2) incubating the sample containing the nucleic acid mixture or libraryand biotinylated probe-target hybridized molecules of prestep (1) in thepresence of a binding ligand of the hapten of the haptenylated probe,the binding ligand being conjugated to support; the incubation beingsufficient to permit the probe molecules, and the probe-targethybridized molecule to become bound to the binding ligand of thesupport;

(3) recovering the probe-target hybridized molecules bound to thesupport from the nucleic acid mixture or library and any unboundbiotinylated probe-target hybridized molecules of prestep (2); and

(4) incubating the recovered support containing the bound probe-targethybridized molecules under conditions sufficient to separate the strandsof double-stranded molecules; the incubation thereby releasing thehybridized target molecule from the biotinylated probe, and generating asample single-stranded desired target molecule for use in step (A).

The invention additionally provides the embodiment wherein thesingle-stranded nucleic acid molecule of the sample contains anucleotide analog, and wherein after completing step A, but prior tocommencing step B, the method additionally comprises the presteps:

(1') incubating the generated double-stranded molecules in the presenceof a nuclease capable of degrading nucleic acid containing nucleotideanalog residues; and

(2') incubating non-degraded nucleic acid with a primer under conditionssufficient to permit the primer to be extended in a template-dependentmanner.

The invention additionally provides the embodiment wherein in step A,the template-dependent extension of the primer is conducted in thepresence of a nuclease resistant nucleotide analog to thereby generate adouble-stranded desired target molecule containing a residue of thenucleotide analog; and wherein prior to commencing the step B, themethod additionally comprises the presteps:

(1") incubating the generated double-stranded desired target molecule inthe presence of a nuclease, wherein the nuclease is substantially unableto cleave a nucleic acid molecule containing the nucleotide analogresidue, but is substantially capable of degrading both single-strandednucleic acid molecules and double-stranded nucleic acid molecules thatlack the nucleic acid analog residue; the incubation being underconditions sufficient to permit such degradation, and therebysubstantially eliminating both single-stranded nucleic acid moleculesand double-stranded nucleic acid molecules that lack the nucleic acidanalog residue from the sample; and thereby forming a preparation havinga substantial enrichment of the desired target molecule relative to theinitial sample; and

(2") recovering the desired molecule from the preparation of prestep(1") to thereby form a library or mixture for the step B. The inventionparticularly concerns the embodiments of the above methods wherein thedesired target nucleic acid molecule is a DNA molecule, asingle-stranded nucleic acid molecule, a circular nucleic acid moleculeand most preferably, a single-stranded, circular DNA molecule.

The invention particularly concerns the embodiments of the above methodswherein the desired target nucleic acid molecule is a DNA or RNAmolecule, a single-stranded nucleic acid molecule, a circular nucleicacid molecule and most preferably, a single-stranded, circular DNAmolecule.

The invention also concerns the embodiments of the above methods whereinthe hapten is biotin, and wherein the binding ligand of the hapten isavidin, streptavidin, or antibody or antibody fragments that bindbiotin.

The invention also concerns the embodiments of the above methods whereinthe support is a bead, especially a paramagnetic bead that can berecovered by magnetic means or other physical separation.

The invention also concerns the embodiments of the above methods whereinthe sequence of the primer molecule may be complementary to the samesequence of the desired target molecule as the probe molecule, or it maybe complementary to a different sequence.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 provides a diagrammatic illustration of a preferred embodiment ofthe isolation method of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns an improved method for rapidly isolatinga "desired" nucleic acid "clone" from a mixture or library of clonedmolecules. The "clones" of the present invention comprise circular orlinear DNA or RNA molecules that may be either single-stranded ordouble-stranded. Typically, such clones or libraries will compriseplasmids or other vectors (such as viral vectors) that have beenengineered to contain a fragment of DNA or RNA derived from a sourcesuch as a homogeneous specimen (such as cells in tissue culture, cellsof the same tissue, etc.), or a heterogeneous specimen (such as amixture of pathogen-free and pathogen-infected cells, a mixture of cellsof different tissues, species, or cells of the same or different tissueat different temporal or developmental stages, etc.). The cells, if any,of these nucleic acid sources may be either prokaryotic or eukaryoticcells (such as those of animals, humans and higher plants).

Various libraries can be selected for large scale preparation. Theconstruction of plasmid, cosmid, and phagemid cDNA libraries, or genomiclibraries are described in Sambrook, J. et al., In: Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)). Preferably, single-stranded phagemid cDNAlibraries can be prepared as described previously by Gruber, C. E. etal., Focus 15: (1993)). The general steps of the method will differdepending upon whether the desired sequence has been cloned intosingle-stranded or double-stranded molecules, and whether such moleculesare DNA or RNA.

As used herein, there is no constraint as to the sequence of the targetnucleic acid molecule whose isolation is desired. Since the presentinvention relies upon nucleic acid hybridization, the target moleculeshould have a length of at least 10 nucleotides in order to beefficiently recovered. No upper limit to the size of the moleculesexists, and the methods of the invention can be used to isolate nucleicacid molecules of several kilobases or more.

The selection method of the present invention is based in part upon theobservation that double-stranded nucleic acid molecules transformbacterial cells with greater efficiency than single-stranded nucleicacid molecules. In one embodiment, the invention achieves the isolationof a desired nucleic acid sequence from a library of sequences byproviding a primer molecule to the mixture. A "primer" or "primermolecule" as used herein is a single-stranded oligonucleotide or asingle-stranded polynucleotide that can be extended by the covalentaddition of nucleotide monomers during the template-dependentpolymerization reaction catalyzed by a polymerase. A primer is typically11 bases or longer; most preferably, a primer is 17 bases or longer.Examples of suitable DNA polymerases include the large proteolyticfragment of the DNA polymerase I of the bacterium E. coli, commonlyknown as "Klenow" polymerase, E. coli DNA polymerase I, thebacteriophage T7 DNA polymerase. Preferably, a thermostable polymerasewill be used, such as a polymerase that can catalyze nucleotide additionat temperatures of between about 50° C. to about 100° C. Exemplarythermostable polymerases are described in European Patent Appln.0258017, incorporated herein by reference. The thermostable "Taq" DNApolymerase (Life Technologies, Inc., Gaithersburg, Md.) is particularlypreferred.

Where the target mixture involved RNA molecules, and a DNA molecule isdesired, a reverse transcriptase may be employed. Reverse transcriptasesare discussed by Sambrook, J. et al. (In: Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)) and by Noonan, K. F. et al. (Nucleic Acids Res.16:10366 (1988)). Similarly, where the target mixture involved RNA, anRNA polymerase may be used. Examples of suitable RNA polymerases includeE. coli RNA polymerase, T7 RNA polymerase, etc.

As a consequence of such polymerization, the desired target molecule,but not other nucleic acid molecules of the mixture, is converted into adouble-stranded form. The mixture can, without further processing, betransformed into suitable recipient bacteria (see, Sambrook, J. et al.,In: Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Transformants can berecovered, and their recombinant DNA or RNA molecules can be extractedand retrieved. Such processing provides a new mixture or library ofnucleic acid molecules that is substantially enriched for the desiredmolecule. Optionally, the above-described method can be repeated (asoften as desired) in order to obtain mixtures or libraries that are morehighly enriched for the desired nucleic acid sequence.

A preferred method for conducting such processing employs a library ormixture of a single-stranded phagemid, such as M13. In such a method, aprimer is used to convert the single-stranded DNA molecule into adouble-stranded form.

A. Capture Enrichment of Desired Molecules

In a first preferred embodiment, the selection method of the presentinvention can be augmented through the use of an optional nucleic acid"capture" step. This embodiment is preferably performed usingsingle-stranded nucleic acid molecules. Where double-stranded circularmolecules are employed, a preferred initial step involves denaturing themolecules into their respective single strands. Most preferably, this isaccomplished by transient incubation of the sample at elevatedtemperatures (60°-80° C. or above the T_(m) of the mixture).Alternatively, salt or ionic conditions can be adjusted, or denaturationcan be accomplished via helicase activity. The strand-separation stepmay require a topoisomerase in order to permit full strand separation.Alternatively, the double-stranded plasmid or linear target DNA could benicked and the nicked strand removed by denaturation or digestion. Inaddition, double-stranded linear DNA could be denatured or one stranddigested. These methods would leave one strand of DNA intact for hybridselection.

In accordance with the invention, such a population of single-strandedmolecules is then incubated in the presence of an oligonucleotide probeunder conditions sufficient to permit and promote sequence-specificnucleic acid hybridization. Hybridization may be conducted underconditions which either permit or minimize random hybridization. As usedherein, conditions which minimize random hybridization are of suchstringency that they permit hybridization only of sequences that exhibitcomplete complementarity. In contrast, conditions that permit randomhybridization will enable molecules having only partial complementarityto stably hybridize with one another. Suitable conditions which eitherpermit or minimize random nucleic acid hybridization are described byManiatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratories, Cold Spring Harbor, N.Y. (1982)), by Haymes,B. D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRLPress, Washington, D.C. (1985), herein incorporated by reference), andsimilar texts.

The probe is a nucleic acid molecule, preferably DNA, preferably greaterthan 8-12 nucleotides in length, and most preferably greater than 15-30nucleotides in length, whose sequence is selected to be complimentary tothe sequence of a region of the target molecule that is to be isolated.The probe thus need not be, and most preferably will not be equal insize to the target molecule that is to be recovered. Two sequences aresaid to be "complementary" to one another if they are capable ofhybridizing to one another to form a stable anti-parallel,double-stranded nucleic acid structure. Thus, the sequences need notexhibit precise complementarity, but need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure. Thus, departures from complete complementarity arepermissible, so long as such departures are not sufficient to completelypreclude hybridization to form a double-stranded structure.

In some embodiments, the sequence of the probe and/or the primer may bederived from amino acid sequence data. In these instances, the probeand/or the primer may have a degenerate sequence. For instance, if onehad an amino acid motif (e.g. zinc fingers) that occurred in a number ofproteins encoded in a library, one could enrich for nucleic acidsencoding proteins having that motif.

In a preferred sub-embodiment, the probe is "haptenylated." As usedherein, a "haptenylated" probe is a nucleic acid molecule that has beencovalently bonded to a hapten molecule. A hapten is a molecule that canbe recognized and bound by another molecule, e.g. an antibody. Examplesof haptens include any antigen, biotin, dinitrophenol, etc. Biotin is apreferred hapten of the present invention and may be bound by proteinssuch as avidin and streptavidin. The probe may be "haptenylated" usingany of a variety of methods well known in the art. For example, methodsfor "biotinylating" the probe are described, for example, by Hevey etal. (U.S. Pat. No. 4,228,237); Kourilsky et al, (U.S. Pat. No.4,581,333); Hofman et al. (J. Amer. Chem. Soc. 100:3585-3590 (1978));Holmstrom, K. et al. (Anal. Biochem. 209:278-283 (1993)), etc. Suchmodification is most preferably accomplished by incorporatingbiotinylated nucleotides into a nucleic acid molecule using conventionalmethods. Alternatively, such modification can be made using photobiotin(Vector Laboratories). Other methods can, of course, be employed toproduce such biotinylated molecules.

The above-described incubation thus results in the hybridization of thebiotinylated probe and the desired target sequence such that adouble-stranded region is formed. In the next step of the preferredmethod, this complex is "captured" using a an immobilizable support,most preferably, a paramagnetic bead. In a preferred sub-embodiment, thecapture of the hybridized biotinylated probe is initiated without thenecessity for removing non-hybridized molecules.

The support is modified so as to have a binding ligand of biotinconjugated to it. Suitable binding ligands include avidin, orstreptavidin, or antibody or antibody fragments that bind biotin.Methods for effecting such covalent attachment are described by Hevey etal. (U.S. Pat. No. 4,228,237) and by Kourilsky et al. (U.S. Pat. No.4,581,333). Suitable solid supports include, but are not limited to,beads, tubes, or plates, which may be made of materials including, butnot limited to, latex, glass, polystyrene, polypropylene or otherplastic. Such supports can be 2-dimensional strips, beads, etc. Apreferred support is a paramagnetic-streptavidin conjugated bead, suchas the Dynabead Streptavidin M-280 bead (Dynal, Great Neck, N.Y.).

The addition of the beads (or other support) to the reaction permits thebiotinylated probe to bind to the avidin (or other biotin ligand) of thebeads. Such binding reactions are very strong. For example, the bindingconstant for the reaction between avidin and biotin is approximately10¹⁵ 1/mole. The very strong nature of this bond has been found topersist even when biotin is conjugated, by means of its carboxyl group,to another molecule, or when avidin is attached to another molecule.

As a consequence of such binding, any biotinylated probe that hashybridized to a desired target molecule will become bound to the bead orsupport. In contrast, non-target molecules will remain unbound, and canbe separated from the bound material by washing, filtration,centrifugation, sieving, or by magnetic separation methods. Mostpreferably, however, a magnet is used to pull the paramagnetic bead outof solution, and the beads are washed with a suitable buffer (such asone containing Tris, EDTA, and NaCl). Such treatment removes themajority of non-target nucleic acid sequences that were originallypresent, and hence eliminates undesired non-selected single-strandedphagemid DNA from the reaction.

The specifically captured single-stranded phagemid target DNA(hybridized to the biotinylated probe) is then released from the probeby treatment such as addition of an alkaline buffer, heat, etc. In apreferred subembodiment, the selected target DNA is resuspended in aformamide-Tris-EDTA buffer and released from the beads by heating at atemperature of 60°-70° C. for a short period of time. The releasingtreatment is preferably selected such that the biotinylated proberemains attached to the magnetic beads, which is then removed fromsolution by sieving, centrifugation, filtration, or more preferably, bya magnet.

B. Nuclease Enrichment of Desired Molecules

In a second preferred embodiment, a nuclease enrichment protocol canoptionally be used to aid, or further aid in effecting the isolation ofa desired target DNA molecule. Where employed, the second embodiment caneither be used alone, or in conjunction with the above-described firstpreferred embodiment.

As in the case of the first preferred embodiment, single-strandednucleic acid is desired. Thus, where double-stranded nucleic acid isemployed, any of the above-described methods can be used to obtain asuitable single-stranded molecule. Such single-stranded molecules areconverted to their double-stranded DNA form using a DNA polymerase andin the presence of requisite nucleotide triphosphates, and factors, andan oligonucleotide primer. Where the first and second embodiments are tobe performed in conjunction, the primer employed in the secondembodiment may comprise all or a subset of the sequence present in thehaptenylated probe molecule of the first embodiment. In such asub-embodiment, therefore, the conversion step need not further sort ordistinguish among the recovered molecules. Preferably, stringenthybridization conditions are used and the conversion step is done athigh temperature with a thermostable polymerase, e.g. Taq polymerase. Inthis case, because the hybridization and conversion are done underconditions favorable to correct hybrids, the conversion step doesfurther enrich or select for the desired target molecule. In analternative sub-embodiment, the primer molecule may comprise a sequencethat although present on the desired target molecule was not containedon the "haptenylated" probe. Such a sub-embodiment permits theexperimentalist to purify only a subset of the initially presentmolecules. For example, if the probe was designed to hybridize to anymember of a gene library that had a particular enhancer sequence, andthe primer were designed to hybridize only to a receptor binding site,the net effect of the reaction would be to obtain double-strandedmolecules that contained both the enhancer sequence and the receptorbinding site.

Note that the hapten need not be covalently coupled to the probe-nucleicacid. The hapten may be linked, either covalently or non-covalently, toa molecule that non-covalently binds the probe molecule, e.g. asingle-stranded DNA binding protein. The binding protein must bindtightly enough that significant quantities of it will not fall off ofthe probe molecules and bind to nucleic acid molecules of the sample.

The template-dependent extension of the primer is conducted in thepresence of at least "nucleotide analog" (either in lieu of or inaddition to the naturally occurring analog). A "nucleotide analog," asused herein, refers to a nucleotide which is not found in the target DNAor RNA that is the primer's template. For example, where the isolatedtarget molecule is DNA, suitable nucleotide analogs includeribonucleotides, deoxyuridine, bromodeoxyuridine, 7-methylguanine,5-methyldeoxcytosine, 5,6-dihyro-5,6-dihydroxydeoxythymidine,3-methyldeoxyadenosine, etc. (see, Duncan, B. K., The EnzymesXIV:565-586 (1981)). Other nucleotide analogs will be evident to thosein the art. Where the template is RNA, deoxynucleotide triphosphates andtheir analogs are the preferred nucleotide analogs. Any single-strandednon-target DNA that remains after the conversion step may contribute abackground of non-target sequences in molecules recovered by the presentmethod, therefore, it is desirable to remove or eliminate anysingle-stranded DNA that might remain.

The presence of the nucleotide analog in the reaction will result in theproduction of a double-stranded molecule that contains incorporatedanalog bases. Such incorporation affects the ability of endonucleasesand exonucleases to cleave or degrade the double-stranded molecule.Thus, in a particularly preferred sub-embodiment, a primer is extendedfrom a circular DNA template in the presence of a methylated nucleotide(for example, 5-methyl dCTP). The resulting double-stranded molecule,which contains incorporated 5-methyl C residues, is resistant tocleavage by many restriction endonucleases. HhaI is particularlypreferred when used in conjunction with 5-methyl C, since it alsodegrades single-stranded DNA, the effect of incubation in the presenceof such enzymes is to destroy most or all residual undesired non-targetmolecules present, and to thereby greatly enrich the concentration ofthe desired vector. Other nucleotide analogs that inhibit or blockexonucleases or restriction endonucleases are 6-methyladenine,5-methylguanine and 5-methylcytidine. Combinations of nucleotide analogsand suitable enzymes are known in the art (see, for example, LifeTechnologies™ 1993-1994 Catalogue and Reference Guide, Chapter 6, LifeTechnologies, Inc., Gaithersburg, Md., herein incorporated byreference).

In a similar manner, where the source library was composed ofsingle-stranded RNA vectors, the use of dNTPs (i.e. dATP, dTTP, dCTP,and dGTP) in the conversion step will render such molecules resistant tomung bean nuclease, or Bal-31 nuclease.

Although the foregoing discussion has emphasized the use of circularmolecules, the methods of the present invention are fully amenable tothe use of linear molecules. In such a case, the primer molecule (butnot necessarily the probe molecule) is preferably selected such that ithybridizes to the 5' terminus of the target molecule. Such selectionwill permit the template-dependent extension of the molecule to producea full length copy of the target molecule.

Desirably, the recovered target DNA is then precipitated with organicsolvents, and resuspended in buffer. The product may then be transfectedor electroporated into recipient cells, for example by the method ofRubenstein et al. (Nucl. Acids Res. 18:4833 (1990), herein incorporatedby reference).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Preferred Method for Isolating Desired Target Molecules

A preferred method for isolating a desired target molecule employs alibrary or mixture of a single-stranded phagemid, such as M13. In such amethod, the single-stranded phagemid is introduced into an ung dutmutant of E. coli (Kunkel, T. A., U.S. Pat. No. 4,873,192; Longo, M. C.et al., Gene 93:125-128 (1990); Hartley, U.S. Pat. No. 5,035,966; allherein incorporated by reference). The "+" strand of phagemids grown insuch mutants contains deoxyuridine (dUTP), and can be recovered from thepackaged virion. Thus, the use of such mutants permits the isolation ofa library or mixture that comprises single-stranded DNA molecules whichcontain dU residues (Kunkel, T. A., U.S. Pat. No. 4,873,192).

The recovered DNA can then be optionally captured via a capture step, ordirectly processed using a nuclease enrichment step.

If a capture step is to be conducted, the dU-containing strands areincubated in the presence of a complementary biotinylated probe. Theprobe, and any hybridized DNA is then recovered by permitting the biotinto bind to avidin-coated paramagnetic beads, and then recovering thebeads from solution using a magnet. The library or mixture is recoveredfrom the beads by denaturation of the hybridized molecules.

The recovered single-stranded DNA is then incubated in the presence of acomplementary primer, dATP, dTTP, dCTP, and dGTP and under conditionssufficient to permit the extension of the primer. Such extension thuscreates a sample that contains single-stranded dU-containing moleculesand double-stranded dU/dT hybrid (desired target) molecules.

Although the triphosphate form of deoxyuridine, dUTP, is present inliving organisms as a metabolic intermediate, it is rarely incorporatedinto DNA. When dUTP is incorporated into DNA, the resulting deoxyuridinecan be promptly removed in vivo by the enzyme uracil DNA glycosylase(UDG) (Kunkel, U.S. Pat. No. 4,873,192; Duncan, B. K., The EnzymesXIV:565-586 (1981), both references herein incorporated by reference intheir entirety).

In this embodiment of the present invention, the mixture of molecules isthen treated, either in vivo or in vitro with UDG. Such treatmentdestroys all of the single-stranded, non-desired, non-target moleculesin the sample. It further destroys the "+" strand of all of thedouble-stranded desired target molecules.

The sample is therefore then either directly transformed into E. coli topermit the isolation of the target molecule or incubated in the presenceof a primer molecule that is capable of hybridizing to the "-" strand ofthe phagemid. Such incubation is under conditions suitable for mediatingthe template-dependent extension of the primer. Hence, such incubationproduces double-stranded molecules that have the sequence of the desiredtarget molecules, and thereby permit the isolation of the targetmolecule.

EXAMPLE 2 Preparation of single-stranded DNA

The large scale preparation of single-stranded phagemid cDNA library wasmade as described previously (Gruber, C. E. et al., Focus 15 (1993),herein incorporated by reference).

Preparation of Biotinylated Oligonucleotides

The oligonucleotide probes were biotin-labeled using biotin-14-dCTP andterminal deoxynucleotidyl transferase (TdT) as described by Flickinger,J. L. et al. (Nucl. Acids Res. 20:2382 (1992)) with the following minormodifications. In a typical reaction, 0.3-0.5 nmol (≈5 μg) ofoligonucleotides (21-25-mer), 500 μM of biotin-14-dCTP and 60 units ofTdT in 50 μl of 1X tailing buffer [100 mM potassium cacodylate (pH 7.2),2 mM CoCl₂ and 200 μM DTT] was incubated at 37° C. for 15 min. Thereaction was terminated by adding 2 μl of 0.25 M EDTA. The labeledprobes were precipitated by adding an equal volume (52 μl) of 1M Trisbuffer (pH 7.5), 10 μg glycogen as carrier, and 2.5 volume (260 μl) ofethanol, and stored on dry ice for 10 min. After centrifugation at 4° C.for 10 min, the probes were rinsed with 100 μl of 75% ethanol andcentrifuged for 2 min. The probes were air-dried and dissolved in 10 μlof TE. To determine the labeling efficiency and the concentration of thelabeled probe, 2 μl of labeled products were resuspended in an equalvolume of sequencing reaction stop buffer [95% (v/v) formamide, 10 mMEDTA (pH 8.0), 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol],heated at 95° C. for 1 min and chilled on ice. The probes wereelectrophoresed along with a known amount of the starting material on16% denaturing PAGE. The gel was soaked in a ethidium bromide solution(0.5 μg/ml) for 15 min, and photographed. Typically, more than 95% ofthe oligonucleotide will be labeled. The concentration of the labeledprobes was determined by the comparison to the known starting material.

Hybrid Selection

The hybridization was performed by the following procedure: 1-10 μg ofsingle-stranded target library DNA was diluted with 10 μl of dilutionbuffer (100 mM HEPES, pH 7.5, 2 mM EDTA and 0.2% SDS) to a final volumeof 19 μl in a 5 ml Falcon tube. The DNA was denatured at 95° C. for 1min and immediately chilled in ice water for 5 min. 1 μl (20 ng) ofbiotin-probe was added to the DNA mixture, followed by the addition of 5μl of 5 M NaCl. The hybridization mixture was incubated at 42° C. withcontinuous shaking (200 rpm) in a culture incubator for 24 h. Beforebinding the hybrids to the streptavidin, 50 μl of the streptavidincoated paramagnetic beads (DYNAL) were washed once with 1X bindingbuffer (10 mM TRIS, pH 7.5, 1 mM EDTA and 1M NaCl) by following themanufacturer's instructions. The paramagnetic beads were resuspended in20 μl of 1X binding buffer. The hybridization mixture was added to theresuspended beads and mixed well. The mixture was incubated at roomtemperature for 1 h with occasional mixing by gently tipping the tube.The paramagnetic beads were separated from the DNA bulk by inserting thetube into the magnet, and washed 6 times with the washing buffer (10 mMTris, pH 7.5, 1 mM EDTA and 500 mM NaCl). Finally, the paramagneticbeads were resuspended in 20 μl of 30% formamide in TE buffer. Theselected DNA was released by heating the beads at 65° C. for 5 min. Thetube was inserted into the magnet, and the aqueous phase was transferredto a new tube. The beads were washed once with 15 μl of TE buffer, andthe aqueous phases were pooled. The selected DNA was precipitated with0.5 volumes of 7.5M ammonium acetate, 10 μg of glycogen, and 2.5 volumeof ethanol. The DNA pellet was dissolved in 5-10 μl of TE buffer. Analiquot (1 μl) was used for the electroporation to determine the hybridselection efficiency.

Repair of Single-Stranded DNA

The remainder of the selected single-stranded DNA was converted todouble-stranded DNA before electroporation as described by Rubenstein etal. (Nucl. Acids Res. 18:4833 (1990)) with some modifications. Thereaction was carried out in 30 μl containing the selectedsingle-stranded DNA, 250 ng of unlabeled primer, 300 μM each dTTP, dGTP,dATP and 5-methyl dCTP, Taq DNA polymerase buffer and 2 units of Taq DNApolymerase. After repair, the mixture was extracted once withphenol:chloroform. The organic phase was back-extracted with 15 μl ofTE, the aqueous phases were pooled and ethanol precipitated. The pelletwas rinsed with 100 μl of 75% ethanol and dried. The repaired DNA wasdissolved in 5-10 μl of TE and digested with HhaI for 2 h at 37° C.After digestion, the mixture was extracted once with phenol:chloroform,ethanol precipitated and dissolved in 5-10 μl of TE.

Detection of the Target Gene

The repaired, digested DNA was used to transform E. coli bacteria (DH10Bbackground) by chemical transformation or electroporation. The targetcolony can be detected by the PCR, colony hybridization or cyclesequencing approach.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A method for obtaining an enriched source of adesired target nucleic acid molecule from an initial sample containing amixture or library of single-stranded nucleic acid containing saiddesired molecule, wherein said method comprises the steps:(A) incubatingsaid initial sample containing said nucleic acid mixture or library inthe presence of a biotinylated nucleic acid probe molecule, said probemolecules having a sequence complementary to a nucleotide sequence ofsaid desired target molecule; said incubating being under conditionssufficient to permit said probe to hybridize to said desired targetmolecule and to thereby generate hybridized molecules wherein saidtarget molecule is bound to said probe; (B) incubating a samplecontaining said hybridized molecules of step (A) in the presence of abiotin-binding ligand immobilized to a support; said incubating beingsufficient to permit said hybridized molecules to become bound to saidbiotin-binding ligand of said support; (C) separating said boundhybridized molecules of step, (B) from Unbound nucleic acid molecules,and recovering said bound hybridized molecules from said support, andthen incubating said recovered hybridized molecules under conditionssufficient to separate the strands of double-stranded nucleic acidmolecules; said incubating thereby releasing said hybridized targetmolecule from said biotinylated probe, and generating single-strandeddesired target molecules; (D) incubating said single-stranded desiredtarget molecules in the presence of a nucleotide analog and a primernucleic acid molecule complementary to a sequence of said desired targetmolecule; wherein said nucleotide analog confers endonuclease resistanceto nucleic acid molecules that contain said analog; said incubatingbeing under conditions sufficient to permit hybridization between saidprimer and said single-stranded desired target molecule, and furthersufficient to permit the template-dependent extension of said primer tothereby generate a double-stranded desired target molecule wherein onestrand of said double-stranded desired target molecule contains saidincorporated nucleotide analog and is resistant to endonuclease; (E)incubating said generated double-stranded molecules in the presence of anuclease capable of degrading nucleic acid that lacks said incorporatedendonuclease-resistant nucleotide triphosphate derivatives, butincapable of degrading nucleic acid that contains said incorporatedendonuclease-resistant nucleotide triphosphate derivatives; wherein saidincubating is conducted under conditions sufficient to degrade nucleicacid that lacks said incorporated endonuclease-resistant nucleotidetriphosphate derivatives to thereby form said enriched source of saiddesired target nucleic acid molecule.
 2. The method of claim 1, whereinin step A said incubating is under conditions which minimize randomhybridization.
 3. The method of claim 1, wherein said desired targetnucleic acid molecule is a DNA molecule.
 4. The method of claim 1,wherein said desired target nucleic acid molecule is an RNA molecule. 5.The method of claim 1, wherein said desired target molecule is acircular nucleic acid molecule.
 6. The method of claim 5, wherein saiddesired target molecule is a circular DNA molecule.
 7. The method ofclaim 1, wherein said biotin-binding ligand is avidin or streptavidin.8. The method of claim 7, wherein said biotin-binding ligand is avidin.9. The method of claim 7, wherein said biotin-binding ligand isstreptavidin.
 10. The method of claim 1, wherein said support is aparamagnetic bead.
 11. The method of claim 10, wherein said hybridizedmolecule bound to said paramagnetic bead is recovered by magnetic means.12. The method of claim 1, wherein said primer molecule of step (D) iscomplementary to the same sequence of said desired target molecule assaid probe molecule of step (A).
 13. The method of claim 1, wherein saidprimer molecule of step (D) is complementary to a sequence of saiddesired target molecule that differs from the sequence of said desiredtarget molecule that is complementary to said probe molecule of step(A).
 14. The method of claim 1, wherein said single-stranded desiredtarget molecule contains deoxyuridine, and wherein said nuclease isuracil DNA glycosylase (UDG).
 15. The method of claim 1, wherein saidnuclease does not cleave hemimethylated DNA.
 16. The method of claim 15,wherein said nucleic acid analog is 5-methylcytidine, and wherein saidnuclease that does not cleave hemimethylated DNA is HhaI.
 17. The methodof claim 1, wherein in step (A), said probe has a degenerate sequence.18. The method of claim 1, wherein in step (D), said primer has adegenerate sequence.
 19. The method of claim 1, wherein said methodadditionally includes the step of amplifying said desired targetmolecule by an in vitro amplification reaction.
 20. The method of claim19, wherein said in vitro amplification reaction is a polymerase chainreaction.